Journal: Cells

Article Title: Development and Validation of AAV-Mediated Liver, Liver-VAT, and Liver-Brain SORT and Therapeutic Regulation of FASN in Hepatic De Novo Lipogenesis

doi: 10.3390/cells14050372

Figure Lengend Snippet: Construction and validation of sc-RNAi vector clones against Creld2 for in vitro knockdown efficiency. ( A ) Vector map of scAAV-H1-RSV-eGFP plasmid showing shRNA cloning sites consisting of BbsI cut site. ( B ) Agarose image -post-scAAV-H1-RSV-eGFP plasmid BbsI-digestion. L, UD, and D1 denote ladder, un-digested, digested 1, respectively. ( C ) shScr clone and five clones of shCreld2 plasmid that target Creld2 coding sequences. The sequences targeting the Creld2 transcript are enlisted below every clone. ( D – F ) N2A cells were transfected with 5 AAV-shCreld2 clones along with AAV-shScr as control. The CRELD2 knockdown efficiency was evaluated using qRT-PCR ( D ), and Western blot analysis ( E ). The CRELD2 band intensity was quantified using Image J, and a graph of the relative expression of (β-ACTIN) was plotted ( F ). Data are shown as mean ± SD (n = 3) and were analyzed in ( D ) using the Kruskal–Wallis test followed by Benjamini and Hochberg multiple comparison test, and in ( F ) using an ordinary one-way ANOVA with Dunnett’s multiple comparison test (ns: non-significant, * p < 0.05, *** p < 0.001).

Article Snippet: Single-stranded shRNA targeting Creld2 and its complementary strand, corresponding to each clone listed in , were dissolved in 1X T4 DNA ligase buffer (NEB, B0202S, Ipswich, MA, USA) to a concentration of one μg/μL.

Techniques: Biomarker Discovery, Plasmid Preparation, Clone Assay, In Vitro, Knockdown, shRNA, Cloning, Transfection, Control, Quantitative RT-PCR, Western Blot, Expressing, Comparison

Journal: Cells

Article Title: Development and Validation of AAV-Mediated Liver, Liver-VAT, and Liver-Brain SORT and Therapeutic Regulation of FASN in Hepatic De Novo Lipogenesis

doi: 10.3390/cells14050372

Figure Lengend Snippet: Whole-body distribution of AAV8-shCreld2 exhibited liver-specific targeting. ( A ) RNA quantification, ( B ) Protein quantification of CRELD2 of liver, colon, kidney, lung, pancreas, spleen, thymus, VAT, muscles, heart, and brain post AAV8-shScr and AAV8-shCreld2 injection. Band intensities were quantified using Image J and plotted relative to GAPDH or β-ACTIN levels. 36b4 was used as a housekeeping gene for all qRT-PCR analyses. Data are represented as mean ± SD (n = 5) and were analyzed using a two-tailed unpaired t test or Mann–Whitney test as appropriate (ns: non-significant, * p < 0.05, *** p < 0.001).

Article Snippet: Single-stranded shRNA targeting Creld2 and its complementary strand, corresponding to each clone listed in , were dissolved in 1X T4 DNA ligase buffer (NEB, B0202S, Ipswich, MA, USA) to a concentration of one μg/μL.

Techniques: Muscles, Injection, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY

Journal: Cells

Article Title: Development and Validation of AAV-Mediated Liver, Liver-VAT, and Liver-Brain SORT and Therapeutic Regulation of FASN in Hepatic De Novo Lipogenesis

doi: 10.3390/cells14050372

Figure Lengend Snippet: Systemic injection of AAV1-shCreld2 did not exhibit knockdown in any of the tested tissues. ( A ) RNA quantification, ( B ) Protein quantification of CRELD2 of liver, colon, kidney, lung, pancreas, spleen, thymus, VAT, muscles, and heart post AAV1-shScr and AAV1-shCreld2 injection. Band intensities were quantified using Image J and plotted relative to GAPDH or β-ACTIN levels. 36b4 was used as a housekeeping gene for all qRT-PCR analyses. Data are represented as mean ± SD (n = 5) and were analyzed using a two-tailed unpaired t test or Mann–Whitney test as appropriate (ns: non-significant, * p < 0.05).

Article Snippet: Single-stranded shRNA targeting Creld2 and its complementary strand, corresponding to each clone listed in , were dissolved in 1X T4 DNA ligase buffer (NEB, B0202S, Ipswich, MA, USA) to a concentration of one μg/μL.

Techniques: Injection, Knockdown, Muscles, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY

Journal: Cells

Article Title: Development and Validation of AAV-Mediated Liver, Liver-VAT, and Liver-Brain SORT and Therapeutic Regulation of FASN in Hepatic De Novo Lipogenesis

doi: 10.3390/cells14050372

Figure Lengend Snippet: AAV5 revealed liver and brain targeting upon systemic distribution. ( A ) RNA quantification, ( B ) Protein quantification of CRELD2 of liver, colon, kidney, lung, pancreas, spleen, thymus, VAT, muscles, heart, and brain post AAV5-shScr and AAV5-shCreld2 injection. Band intensities were quantified using Image J and plotted relative to GAPDH levels. 36b4 was used as a housekeeping gene for all qRT-PCR analyses. Data are represented as mean ± SD (n = 4–5) and were analyzed using the two-tailed unpaired t test or Mann–Whitney test as appropriate (ns: non-significant, * p < 0.05, ** p < 0.01).

Article Snippet: Single-stranded shRNA targeting Creld2 and its complementary strand, corresponding to each clone listed in , were dissolved in 1X T4 DNA ligase buffer (NEB, B0202S, Ipswich, MA, USA) to a concentration of one μg/μL.

Techniques: Muscles, Injection, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY

Journal: Cells

Article Title: Development and Validation of AAV-Mediated Liver, Liver-VAT, and Liver-Brain SORT and Therapeutic Regulation of FASN in Hepatic De Novo Lipogenesis

doi: 10.3390/cells14050372

Figure Lengend Snippet: AAV7 displayed liver and VAT targeting upon systemic distribution. ( A ) RNA quantification, ( B ) Protein quantification of CRELD2 of liver, colon, kidney, lung, pancreas, spleen, thymus, VAT, muscles, and heart post AAV7-shScr and AAV7-shCreld2 injection. Band intensities were quantified using Image J and plotted relative to GAPDH levels. 36b4 was used as a housekeeping gene for all qRT-PCR analyses. Data are represented as mean ± SD (n = 4) and were analyzed using the two-tailed unpaired t test or Mann–Whitney test as appropriate (ns: non-significant, * p < 0.05, ** p < 0.01, **** p < 0.0001).

Article Snippet: Single-stranded shRNA targeting Creld2 and its complementary strand, corresponding to each clone listed in , were dissolved in 1X T4 DNA ligase buffer (NEB, B0202S, Ipswich, MA, USA) to a concentration of one μg/μL.

Techniques: Muscles, Injection, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY

Journal: Cells

Article Title: Development and Validation of AAV-Mediated Liver, Liver-VAT, and Liver-Brain SORT and Therapeutic Regulation of FASN in Hepatic De Novo Lipogenesis

doi: 10.3390/cells14050372

Figure Lengend Snippet: Bio-functional evaluation of AAV8-shCreld2 in models of dyslipidemia. ( A ) CRELD2 , FASN gene expression along with their correlation analysis in the liver of healthy (n = 5) vs. NASH (n = 12) human patients. p -value < 0.05 with the Benjamini and Hochberg method. The ratio of CRELD2 and FASN was plotted in log scale. ( B ) Creld2 , Fasn gene expression along with their correlation analysis in the liver of mice fed with standard chow diet vs. NASH diet (n = 4–5). Ratio of Creld2 and Fasn was plotted in log scale. ( C ) Creld2 expression analysis both at transcript and protein levels with densiometric quantification in liver of mice that were overnight fasted vs. refed for 1 h (n = 4). ( D ) Creld2 , Fasn gene expression along with their correlation analysis in liver of AAV8-shScr vs. AAV8-shCreld2 mice that were 1 h fed after overnight fasting (n = 4). Ratio of Creld2 and Fasn was plotted. Data are represented as mean ± SD and were analyzed using the two-tailed unpaired t test or Mann–Whitney test, Pearson or Spearman correlation analysis as appropriate (ns: non-significant, * p < 0.05, ** p < 0.01, **** p < 0.0001).

Article Snippet: Single-stranded shRNA targeting Creld2 and its complementary strand, corresponding to each clone listed in , were dissolved in 1X T4 DNA ligase buffer (NEB, B0202S, Ipswich, MA, USA) to a concentration of one μg/μL.

Techniques: Functional Assay, Gene Expression, Expressing, Two Tailed Test, MANN-WHITNEY